Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.
Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.
D000596:Amino Acids / Q000032:analysis*; D002268:Carboxypeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005934:Glucagon / Q000378:metabolism; D007202:Indicators and Reagents; D008722:Methods; D010455:Peptides / Q000378:metabolism*; D013997:Time Factors; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D000596:Amino Acids; D007202:Indicators and Reagents; D010455:Peptides; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D005934:Glucagon; D004124:p-Dimethylaminoazobenzene; D002268:Carboxypeptidases; C022633:serine carboxypeptidase